AAV vectors are among a small number of recombinant virus vector systems which have been shown to be useful as in vivo gene transfer agents (reviewed in Carter, 1992, Curr. Opin. Biotech., 3:533-539; Muzyczka, 1992, Curr. Top. Microbiol. Immunol. 158:97-129) and thus are potentially of great importance for human gene therapy. AAV vectors are capable of high-frequency stable DNA integration and expression in a variety of cells, including cystic fibrosis (CF) bronchial and nasal epithelial cells (see, e.g., Flotte et al., 1992, Am. J. Respir. Cell Mol. Biol. 7:349-356; Egan et al., 1992, Nature, 358:581-584; Flotte et al., 1993a, J. Biol. Chem. 268:3781-3790; and Flotte et al., 1993b, Proc. Natl. Acad. Sci. USA, 93:10163-10167); human bone marrow-derived erythroleukemia cells (see, e.g., Walsh et al., 1992, Proc. Natl. Acad. Sci. USA, 89:7257-7261); and several others. Unlike retroviruses, AAV does not appear to require ongoing cell division for stable integration; a clear advantage for gene therapy in tissue such as the human airway epithelium where most cells are terminally differentiated and non-dividing.
AAV is a defective parvovirus that generally replicates only in cells in which certain functions are provided by a co-infecting helper virus. General reviews of AAV may be found in Carter, 1989, Handbook of Parvoviruses, Vol. I, pp. 169-228; and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. Examples of co-infecting viruses that provide helper functions for AAV growth and replication are adenoviruses, herpesviruses and, in some cases, poxviruses such as vaccinia. The nature of the helper function is not entirely known but it appears that an indirect effect of the helper virus is to render the cell permissive for AAV replication. This belief is supported by the observation that in certain cases AAV replication may occur at a low level of efficiency in the absence of helper virus co-infection if the cells are treated with agents that are genotoxic or that disrupt the cell cycle.
Generally, in the absence of helper virus, AAV infection results in high-frequency, stable integration of the AAV genome into the host cell genome. The integrated AAV genome can be rescued and replicated to yield a burst of infectious progeny AAV particles if cells containing an integrated AAV provirus are superinfected with a helper virus such as adenovirus. Since the integration of AAV appears to be an efficient event, AAV can be a useful vector for introducing genes into cells for stable expression for uses such as human gene therapy.
AAV has a very broad host range without any obvious species or tissue specificity and will replicate in virtually any cell line of human, simian or rodent origin, provided that an appropriate helper is present. AAV appears to be ubiquitous as it has been isolated from a wide variety of animal species, including most mammalian and several avian species.
AAV has not been associated with the cause of any disease and AAV is not a transforming or oncogenic virus. AAV integration into chromosomes of human cell lines does not cause any significant alteration in the growth properties or morphological characteristics of the cells. These properties of AAV further recommend it as a potentially useful human gene therapy vector because most of the other viral systems proposed for this application (such as retroviruses, adenoviruses, herpesviruses, or poxviruses) are disease-causing viruses.
AAV particles are comprised of a capsid having three proteins, VP1, VP2, and VP3, and enclosing a DNA genome. The AAV DNA genome is a linear single-stranded DNA molecule having a molecular weight of about 1.5×106 daltons or approximately 4680 nucleotides long. Strands of either sense (“plus” or “minus”) are packaged into individual particles but each particle has only one DNA molecule. Equal numbers of AAV particles contain either a plus or minus strand. Virus particles containing either strand are equally infectious and replication occurs by conversion of the parental infecting single stranded DNA to a duplex form and subsequent amplification of a large pool of duplex molecules from which progeny single strands are displaced and packaged into capsids. Duplex or single-strand copies of AAV genomes inserted into bacterial plasmids or phagemids can result in infectious particles when transfected into adenovirus-infected cells, and this has allowed the study of AAV genetics and the development of AAV vectors.
In the case of subtype AAV2, the genome has two copies of a 145-nucleotide-long ITR (inverted terminal repeat), one on each end of the genome, and a unique sequence region of about 4470 nucleotides long (Srivastava et al., 1983, J. Virol., 45:555-564) that contains two main open reading frames for the rep and cap genes (Hermonat et al., J. Virol. 51:329-339; Tratschin et al., 1984a, J. Virol., 51:611-619). The unique region contains three transcription promoters, p5, p19, and p40, that are used to express the rep and cap genes. Laughlin et al., 1979, Proc. Natl. Acad. Sci. USA, 76:5567-5571.
ITR sequences are involved in a variety of activities in the AAV life cycle. The ITR sequences, each of which can form a hairpin structure, provide a functional origin of replication (ori) and are required in cis for AAV DNA replication and for rescue and excision from prokaryotic plasmids (Samulski et al., 1983, Cell 33: 135-143; Samulski et al., 1987, J. Virol. 61: 3096-3101; Senapathy et al., 1984, J. Mol. Biol. 179: 1-20; Gottlieb and Muzyczka, 1988, Mol. Cell. Biol. 6: 2513-2522). In addition, the ITRs appear to be the minimum sequences required for AAV proviral integration and for packaging of AAV DNA into virions (McLaughlin et al., 1988, J. Virol. 62: 1963-1973; Samulski et al., 1989, J. Virol. 63: 3822-3828; Balague et al., 1997, J. Virol. 71: 3299-3306). In the case of DNA replication, it is clear that most of the terminal 125 nucleotide palindrome is required for DNA replication and terminal resolution (Bohenzky et al., 1988, Virology 166: 316-327; LeFebvre et al., 1984, Mol. Cell. Biol. 4:1416-1419; Im and Muzyczka, 1989, J. Virol. 63: 3095-3104; Ashktorab and Srivastava, 1989, J. Virol. 63: 3034-3039).
Several reports indicated that ITRs generally do not behave as transcriptional regulatory sequences (Muzyczka, 1992; and Walsh et al., 1992) and the deletion of the ITR does not have a major effect on AAV p5 promoter activity (Flotte et al., 1992). Since ITRs were not thought to provide transcriptional activity, AAV vectors have been constructed using AAV promoters to express heterologous genes. See, for example, Carter et al., U.S. Pat. No. 4,797,368, issued Jan. 10, 1989. Subsequent reports by Carter and collaborators have shown ITRs to have a low amount of transcriptional activity in transient and stable expression assays. See, e.g., Carter et al. U.S. Pat. No. 5,587,308, issued Dec. 24, 1996, and Flotte et al., 1993a.
In addition to the requirement that ITR sequences be present in cis, the AAV rep and cap genes are required, in cis or in trans, to provide functions for the replication and encapsidation of the viral genome, respectively. As described below, recombinant AAV (rAAV) vectors for use in gene therapy preferably do not contain the AAV cap or rep genes, but rather these genes can be provided by a host cell used for packaging (typically referred to as an “AAV producer cell”).
In the intact AAV genome, the rep gene is expressed from two promoters, p5 and p19, as noted above. Transcription from p5 yields an unspliced 4.2 kb mRNA which encodes a nonstructural protein, Rep78, and a spliced 3.9 kb mRNA which encodes a second nonstructural protein, Rep68. Transcription from p19 yields an unspliced mRNA which encodes Rep52 and a spliced 3.3 kb mRNA which encodes Rep40. Thus, the four Rep proteins all comprise a common internal region sequence but differ in their amino and carboxyl terminal regions. Only Rep78 and Rep68 are required for AAV duplex DNA replication, but Rep52 and Rep40 appear to be needed for progeny, single-strand DNA accumulation. Mutations in Rep78 and Rep68 are phenotypically Rep(−) whereas mutations affecting only Rep52 and Rep40 are Rep(+) but Ssd(−). Rep68 and Rep78 bind specifically to the ITR at sites known as RRS (Rep recognition sequences) or RBS (Rep binding sites) and the proteins possess several enzymatic activities required for resolving replication at the AAV termini. Rep52 and Rep40 have none of these properties.
The Rep proteins, primarily Rep78 and Rep68, exhibit several pleiotropic regulatory activities, including positive and negative regulation of AAV gene expression and expression from some heterologous promoters, as well as inhibitory effects on cell growth (Tratschin et al., 1986, Mol. Cell. Biol. 6:2884-2894; Labow et al., 1987, Mol. Cell. Biol., 7:1320-1325; Khleif et al., Virology, 181:738-741). The AAV p5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., 1986). Perhaps because of the inhibitory effects of expression of rep on cell growth, constitutive expression of rep in cell lines has not been readily achieved. For example, Mendelson et al. (1988, Virology, 166:154-165) reported a very low level expression of some Rep proteins in certain cell lines after stable integration of AAV genomes.
The structural proteins VP1, VP2, and VP3 all share a common overlapping sequence but differ in that VP1 and VP2 contain additional amino terminal sequences. All three are coded from the same cap gene reading frame expressed from a spliced 2.3 kb mRNA transcribed from the p40 promoter. VP2 and VP3 are generated from the same mRNA by use of alternate initiation codons. VP1 is encoded by a minor mRNA using a 3′ donor site that is 30 nucleotides upstream from the 3′ donor used for the major mRNA that encodes VP2 and VP3. VP1, VP2, and VP3 are all required for capsid production. Mutations which eliminate all three proteins (Cap(−)) prevent accumulation of single-strand progeny AAV DNA whereas mutations in the VP1 amino-terminus (Lip(−), Inf(−)) permit single-strand production but prevent assembly of stable infectious particles.
The genetic analysis of AAV described above was in large part based upon mutational analysis of AAV genomes that were molecularly cloned into bacterial plasmids. In early work, molecular clones of infectious genomes of AAV were constructed by inserting double-strand molecules of the AAV genome into plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. Sci. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 259:4661-4666). Transfection of such AAV recombinant plasmids into mammalian cells that were also infected with an appropriate helper virus, such as adenovirus, could result in rescue and excision of the AAV genome free of any plasmid sequence, replication of the rescued genome, and generation of progeny infectious AAV particles. This provided the basis for performing genetic analysis of AAV as summarized above and permitted construction of AAV transducing vectors.
Based on the genetic analysis, the general principles of AAV vector construction were defined (for reviews, see, e.g., Carter, 1992; Muzyczka, 1992). rAAV vectors can be constructed in AAV recombinant plasmids by substituting portions of the AAV coding sequence with foreign DNA to generate a vector plasmid. In the vector plasmid, the terminal ITR portions (ITRs) of the AAV genome must be retained because of their aforementioned role in excision from the plasmid after transfection, replication of the vector genome and integration and rescue from a host cell genome. The vector can then be packaged into an AAV particle to generate an AAV transducing virus, by transfection of the vector plasmid into cells that are infected by an appropriate helper virus, such as adenovirus or herpesvirus. In order to achieve replication and encapsidation of the vector genome into AAV particles, the vector plasmid must be complemented in trans for any AAV functions, namely rep and cap, that were deleted in construction of the vector plasmid.
Several systems of using rAAV vectors to package foreign DNA and transduce it into various cells have been described. The first rAAV vectors that were described contained foreign reporter genes such as neo, cat or dhfr that were expressed from AAV transcription promoters or an SV40 promoter (Tratschin et al., 1984b, Mol. Cell. Biol. 4:2072-2081; Hermonat and Muzyczka, 1984, Proc. Natl. Acad. Sci. USA, 81:6466-6470; Tratschin et al., 1985, Mol. Cell. Biol. 5:3251-3260; McLaughlin et at., 1988, J. Virol., 62:1963-1973; Lebkowski et at., 1988 Mol. Cell. Biol. 8:3988-3996). These vectors were packaged into AAV-transducing particles by co-transfection into adenovirus-infected cells together with a second packaging plasmid that contained the AAV rep and cap genes expressed from the wild-type AAV transcription promoters.
Samulski et al. (1987) constructed a plasmid, pSub201, which was an intact AAV genome in a bacterial plasmid but which had a deletion of 13 nucleotides at the extremity of each ITR and thus, was rescued and replicated less efficiently than other AAV plasmids that contained the entire AAV genome. Samulski et al. (1989) constructed other vectors based on pSub201 but deleted for rep and cap and containing either a hyg or neo gene expressed from an SV40 early gene promoter. These vectors were packaged into viral particles by co-transfection with a packaging plasmid called pAAV/Ad which consisted of the entire AAV nucleotide sequence from nucleotide 190 to 4490, enclosed at either end with one copy of an adenovirus 5 terminal repeat. In this packaging plasmid, the AAV rep and cap genes were expressed from the wild-type AAV promoters p5, p19, and p40. Since it is missing the ITRs, the AAV genome of pAAV/Ad does not appear to replicate.
Several other reports have described rAAV vectors. Srivastava et al. (1989, Proc. Natl. Acad. Sci. USA, 86:8078-8082) described an AAV vector, based on the pSub201 plasmid of Samulski et al. (1987), in which the coding sequences of AAV were replaced with the coding sequences of another parvovirus, B19. Since this system was based on pSub201 and it suffers from the defect described above for the pSub201 plasmid. Also, the vector and the packaging plasmid both contained the same ITR regions and thus recombination to give contaminating wild-type virus was highly likely. Chatterjee et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 85-89), Wong et al. (1991, Vaccines 91, Cold Spring Harbor Laboratory Press, pp. 183-189), and Chatterjee et al. (1992, Science, 258:1485-1488) describe rAAV vectors designed to express antisense RNA directed against infectious viruses such as HIV or Herpes simplex virus. Other reports have described the use of rAAV vectors to express genes in human lymphocytes (Muro-Cacho et al., 1992, J. Immunotherapy, 11:231-237) and in a human erythroid leukemia cell line (Walsh et al., 1992) with vectors based on the pSub201 vector plasmid and pAAV/Ad packaging plasmid.
Transduction of human airway epithelial cells, isolated from a cystic fibrosis patient and grown in vitro, with a rAAV vector expressing the selective marker gene neo from the AAV p5 promoter was achieved (Flotte et al., 1992). In this study, the AAVneo vector was packaged into AAV particles using the pAAV/Ad packaging plasmid.
The above-cited studies suggest that rAAV vectors may have potential utility as vectors for treatment of human disease by gene therapy. However, a severe limitation on the development of human gene therapy using rAAV vectors has been the inability to efficiently package long pieces of transgene DNA into viral capsids and to effectively express them in recipient cells. Other viral vectors, including, for example, Adenoviral vectors, also exhibit packaging size constraints, however, AAV appears to be particularly sensitive with respect to size constraints. In particular, as the optimal size is exceeded, there is a sharp and dramatic drop-off in vector production.
AAV can package a genome slightly larger than the size of a wild-type genome (about 4.6 kb). The precise relationship of genome size and efficiency of packaging has only recently been defined. Using a series of rAAV vectors with progressively increasing genome lengths, from 1.9 to 6.0 kb, Dong et al. (1996, Human Gene Ther. 7: 2101-2112) were able to analyze quantitatively the packaging efficiency of rAAV in relation to the vector size and to determine the size limit for packaging. Specifically, the packaging efficiencies of rAAV vectors of various sizes were determined directly by assaying DNA contents of viral particles, and indirectly by analyzing their transfer of a chloramphenicol acetyltransferase (CAT) reporter gene into target cells. Dong et al. (1996) showed that the optimal size of an rAAV vector for packaging is between 4.1 and 4.9 kb. Although AAV can package a vector larger than its genome size, including vectors up to about 5.2 kb, the packaging efficiencies in this large size range were sharply reduced. When the AAV genome size was smaller than 4.1 kb, the packaging efficiency was also suboptimal. When the size of the genome was less than half the length of the wild-type genome, two copies of the vector were packaged into each virion, suggesting that the copy number control during packaging is a “head-full” mechanism.
Dong et al. (1996) co-transfected the rAAV vectors of various sizes and the pAAV/Ad packaging plasmid (Samulski et al., 1989), into HeLa cells. AAV virions produced from the transiently transfected cells were collected and used to infect fresh HeLa cells; CAT activities in the infected cells were analyzed at 3 days post infection. The resultant CAT activity of vectors from 3.2 to 4.88 kb in length ranged from 80.7 to 129.5 cpm. However, with only a 0.2 kb increase in size beyond 4.88 kb, the resultant CAT activity dropped to 35.9 cpm, indicating a greater than 50% decrease in particle production. Further increases in size resulted in even greater decreases in particle packaging efficiency.
In sum, while recombinant rAAV vectors are believed to have utility for gene therapy, a significant obstacle has been the limitation in the amount of transgene DNA which can be efficiently packaged into viral capsids and then expressed in the recipient cells. This is a particular problem for in vivo applications which require the transfer of larger genes.
While many genes, including their native or a heterologous promoter, are small enough to fit within the size constraints of AAV packaging vectors, many others are not.
One approach to accommodate the AAV packaging constraints is to forego the use of an exogenous transcriptional promoter. In the case of the cystic fibrosis transmembrane conductance regulator (CFTR), for example, it has been shown that, even without any additional promoter, it is possible to construct and use rAAV-CFTR vectors based on the relatively low-level transcriptional activity provided by the AAV ITR itself as described by Carter and collaborators (U.S. Pat. No. 5,587,308; Flotte et al., 1993a).
Another approach is to employ transgenes which have had non-essential coding regions deleted. For example, as described by Carter et al., truncated CFTR genes in recombinant rAAV vectors have been packaged into AAV particles and used to complement the CF defect in mammalian cells. See Carter et al. U.S. patent application Ser. No. 08/455,552, now proceeding to issuance.
The aforementioned approaches exemplified by Carter et al. with regard to the CFTR gene have been quite useful and have effectively enabled the generation of rAAV vectors for use in gene therapy to treat diseases such as cystic fibrosis. Indeed, the success with these approaches has merited the initiation of two different clinical trials involving cystic fibrosis patients being sponsored by Targeted Genetics Corporation at several centers including Stanford University School of Medicine, Stanford, Calif., Johns Hopkins Children's Center, Baltimore, Md., and University of Florida, Gainesville, Fla.
There is, however, a continuing desire for improved rAAV constructs in which transgene expression can be further elevated, despite potential vector size constraints. It would be most useful to have modified rAAV vectors that provide for high efficiency particle production and enhanced expression of inserted transgenes. The present invention provides transcriptionally-activated rAAV vectors that can be employed in these contexts.